戊型肝炎病毒一步法多重荧光定量PCR分型鉴定技术的建立及应用  

Development of one-step multiplex real time RT-PCR for detection and typing of hepatitis E virus genotypes

作　　者：李爱云[1] 帅江冰 曾若雪 孙涛[3] 鱼海琼[4] 朱道中[4] 朱宇宁[1] 张晓峰 李肖梁[5] LI Ai-yun;SHUAI Jiang-bing;ZENG Ruo-xue;SUN Tao;YU Hai-qiong;ZHU Dao-zhong;ZHU Yu-ning;ZHANG Xiao-feng;LI Xiao-liang(Women's Hospital,School of Medicine,Zhejiang University,Hangzhou 310006,China;Zhejiang Academy of Science and Technology for Inspection and Quarantine,Hangzhou 310016,China;Institute of Inspection and Quarantine,Shandong Entry-Exit Inspection and Quarantine Bureau,Qingdao 266500,China;Institute of Inspection and Quarantine,Guangdong Entry-Exit Inspection and Quarantine Bureau,Guangzhou 510623,China;Institute of Preventive Veterinary Medicine,Zhejiang University,Hangzhou 310058,China)

机构地区：[1]浙江大学医学院附属妇产科医院,浙江杭州310006 [2]浙江省检验检疫科学技术研究院,浙江杭州310016 [3]山东出入境检验检疫局检验检疫技术中心,山东青岛266500 [4]广东出入境检验检疫局检验检疫技术中心,广东广州510623 [5]浙江大学动物预防医学研究所,浙江杭州310058

出　　处：《中国预防兽医学报》2018年第11期1020-1025,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：浙江省重点研发计划(2015C02044);国家质检总局科技计划(2013IK046);浙江出入境检验检疫局科技计划(2016-ZKZ-02)

摘　　要：为建立鉴别检测戊型肝炎病毒(HEV)各基因型的方法,本研究基于HEV1、HEV3和HEV4的基因组序列,设计一对保守扩增引物和3条型特异性探针,通过体系优化,建立了用于鉴别检测不同型HEV感染的多重荧光定量PCR方法,并以绿色荧光蛋白(EGFP)为内部质控,确保整个诊断流程的准确和有效。特异性试验结果显示该方法仅对HEV扩增为阳性,而对甲型肝炎病毒、乙型肝炎病毒、疱疹病毒、猪瘟病毒、猪流行性腹泻病毒等核酸扩增均为阴性,特异性好。敏感性试验结果显示,该方法对HEV 1、HEV3和HEV 4的最低检测限均为50个基因组拷贝/反应,敏感性高。分析表明一步法多重荧光定量PCR对不同浓度的HEV 1、HEV3和HEV4重复性试验的变异系数均小于4%,表明该方法具有良好的检测重复性和稳定性。对采集自浙江、安徽、江西、上海和福建省等地区的猪粪便、猪血清和人血清等临床样品进行检测,其结果与传统套式PCR及测序的鉴定结果一致,表明该多重荧光定量PCR对于临床样品的检测具有高准确性,可用于临床HEV隐性感染或早期感染的快速检测。本研究为该病的有效治疗奠定基础。In this study,a multiplex real-time reverse transcription polymerase chain reaction (RT-qPCR)combining a pair of genotype-conserved primer with three specific TaqMan-probes for hepatitis E virus 1(HEVI),HEV3and HEV4,respectively,was developed for monitoring and typing of HEV infection.In the reaction,an in vitro transcript of the enhanced green fluorescent protein (EGFP)gene was introduced as an intemal control to provide a reliable and simple way of monitoring both the sample manipulation and amplification procedures.The multiplex RT-qPCR assay showed a high analytical sensitivity of less than 50 copies RNA per reaction for all HEV genotypes.The specificity and amplification efficiency of the multiplex assay for the respective HEV were confirmed by co-amplification of the other target including hepatitis A virus,hepatitis B virus,herpesvirus as well as swine fever virus,porcine epidemic diarrhea vires.By comparing with the results of nested PCR and sequencing,HEV infection in a panel of clinical samples was reliably detected and typed,indicating that the established multiplex RT-qPCR assay could be used for sensitive detection and rapid differentiation of HEV genotype 1,3 and 4.

关 键 词：戊型肝炎病毒 基因型 荧光定量PCR 多重 

分 类 号：S852.65[农业科学—基础兽医学]